Receptor tyrosine kinase signaling and other receptor mediated signaling pathways typically progress through a pathway that includes ligand binding, receptor activation, phosphorylation of signaling intermediary proteins in the cytoplasm that amplify or network the signal, translocation of a subset of the signaling proteins into the nucleus, followed by activation of transcription, by either the translocated protein or other nuclear co-factors.
EGFR (HER1) is a member of the HER family of receptor tyrosine kinases (RTKs). Each RTK has a ligand-binding domain, a single membrane-spanning region, and a cytoplasmic tyrosine-kinase-containing domain. Under normal physiological conditions, activation typically is controlled by both the temporal and spatial expression of the RTK ligands including mechanisms such as homo- or hetero-dimerization of receptors, activation of kinase domains, phosphorylation, and resulting docking sites in modified domains that allow for activation of downstream signaling pathways involved in growth, proliferation, and/or survival such as Ras/MAPK, PI3K/Akt, PLCγ, and STAT. Dysregulation of the EGFR/HER1 signaling pathway is known to be involved in the development and growth of many tumors, including bladder, brain, breast, colon, esophagus, head, kidney, lung, ovary, neck, pancreas, prostate and stomach. (See e.g., Marmor, M. D., K. B. Skaria, and Y. Yarden. 2004. Signal transduction and oncogenesis by ErbB/HER receptors Int. J. Radiat. Oncol. Biol. Phys. 58:903-913; Olayioye, M. A., R. M. Neve, H. A. Lane, and N. E. Hynes. 2000. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J. 19:3159-3167; Riese, D. J., and D. F. Stern. 1998. Specificity within the EGF family/ErbB receptor family signaling network. Bioessays. 20:41-48; Schlessinger, J. 2004. Common and distinct elements in cellular signaling via EGF and FGF receptors. Science. 306:1506-1507; and Yarden, Y., and M. X. Sliwkowski. 2001. Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2:127-137.)
The monoclonal antibody Erbitux (cetuximab), which targets EGFR, has FDA approval for the treatment of colorectal cancer. In addition, Iressa and Tarceva, two small molecules that target EGFR, have FDA approval for non small cell lung cancer. DakoCytomation California, Inc. provides an FDA approved companion diagnostic that can detect EGFR in patient biopsies. This test can be useful, for example to identify patients, who should respond favorably to EGFR targeted therapeutics. However, experience has shown that currently available EGFR diagnostic tests do not correlate well with patient response to treatment with and EGFR targeted therapeutic.
US2006/0094068 and US2007/0059785 each teach an assay for one or another of a molecular marker (which may include pERK or a total cytoplasmic ERK compared to total nuclear ERK) that may be useful in predicting if an individual will or determining if an individual patient is responding to treatment with an EGF or EGFR inhibitor. The method requires a pre and post treatment biopsy from a subject treated with an EGF or EGFR inhibitor.
There is a need for methods for determining the signal transduction pathway activation state with specific reference to the receptor of interest which likely requires that the receptor and pathway effector molecule expression levels be determined in the same assay to achieve adequate levels of accuracy.
There is also a need for methods for determining the signal transduction pathway activation state associated with a receptor that evaluates more than one possible pathway in the same assay.
There is also a need for an assay that does not require a post treatment biopsy. The lability of phosphorylated proteins, even during short ischemic times between tissue excision and fixation (or freezing) may limit the clinical utility of an assay based strictly on detection of phosphorylated proteins. Therefore there is also a need to devise alternative strategies for determining signal transduction pathway activation state without relying on accurate detection of phosphorylated effector molecules, especially for those molecules that turn out to be unstable in routine clinical practice or when suitably specific detection reagents are not available.
There is also a need for a method that can provide prognostic information based on the receptor expression and the signal transduction pathway(s) activation state.